Transduction of mechanical stimuli - like those of sound and head movements - is the fundamental role of the inner ear and its sensory hair cells. Hair cells transduce deflections of their hair bundles, the sensory organelle, with stunning sensitivity. While well described biophysically, the molecular composition and dynamics of the hair bundle's mechanotransduction apparatus are still mostly mysterious. The goal of our project is to identify key molecules involved in mechanotransduction, then to determine how they interact to form the transduction apparatus. We use a multidisciplinary approach that emphasizes biochemical techniques, including purification of hair bundles and detection of specific proteins by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). In addition, we transfect hair cells with key bundle proteins or their fragments, for example tagged with fluorescent proteins, to allow us to test their roles in transduction or localize them without using antibodies. In the proposed project, we will determine how five key hair-bundle components form the transduction apparatus: (1) the bundle actin- based cytoskeleton;(2) myosin-1c, the adaptation-motor myosin;(3) the tip link, hypothesized to be composed of cadherin 23 and protocadherin 15;(4) the transduction channel, thought to be a member of the transient receptor potential (TRP) family;and (5) the bundle Ca2+ pump, plasma-membrane Ca2+- ATPase isoform 2. These molecules and others we expect to identify together form a transducer that can detect hair-bundle displacements of less than a nanometer, arising from sounds so faint that they are nearly drowned out by air molecules randomly impacting the ear drum and water molecules randomly deflecting bundles. Lay summary. Mechanotransduction by hair cells is a central event in hearing;disruption of transduction leads to profound deafness. Hair cells are the sensory cells of the inner ear;they detect and encode (transduce) sound. The complex of proteins in hair cells that transduce sound are still largely unknown;the goal of this project is to identify and characterize them, as they are essential for hearing.